title: Activation of endothelial cells induces platelet thrombus formation on their matrix. Studies of new in vitro thrombosis model with low molecular weight heparin as anticoagulant.
abstract: Previous studies have indicated that activation of endothelial cells may lead to the production of tissue factor. We have studied the effect of endothelial cell activation and subsequent tissue factor synthesis on thrombus formation on the extracellular matrix in flowing blood. Endothelial cells were stimulated with tumor necrosis factor, endotoxin, or phorbol ester. Coverslips with activated cells or their extracellular matrix were introduced into a perfusion system and exposed to blood anticoagulated with 20 U/ml low molecular weight heparin. This concentration allowed manipulation of blood without activation of the coagulation cascade. Platelet deposition and fibrin formation were evaluated by morphometry, and fibrinopeptide A formation was assayed as a measure of thrombin generation. Activation of endothelial cells caused fibrinopeptide A generation in the perfusate and some deposition of fibrin on endothelial cells; however, platelets were not deposited. The matrix of the stimulated endothelium also caused enhanced fibrinopeptide A generation, and platelet aggregates and fibrin were deposited on the matrix. Maximal effects were observed with stimulation periods between 4 and 10 hours and were still clearly present after 18 hours. Increase in shear rate, perfusion time, and platelet number resulted in an increase in platelet adhesion, but platelet aggregate formation as a percentage of adhesion remained constant. Platelet aggregate formation and fibrinopeptide A generation were inhibited with antibodies against tissue factor or factor VIIa. Platelet aggregate formation alone was inhibited by antibodies against glycoprotein IIb/IIIa. Polymerization of fibrin on the matrix was best supported in perfusions at a low shear rate. The new in vitro thrombosis model presented here provides a powerful tool for study of the regulation of thrombogeneity by the vessel wall in response to various stimuli.
authors.affiliation: Department of Hematology, University Hospital, Utrecht, The Netherlands.
authors.forename: J J
authors.initials: JJ
authors.lastname: Zwaginga
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: P G
authors.initials: PG
authors.lastname: de Groot
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D000937:Antigen-Antibody Reactions; D001777:Blood Coagulation; D001779:Blood Coagulation Factors / Q000494:pharmacology; D001792:Blood Platelets / Q000502:physiology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005109:Extracellular Matrix / Q000166:cytology; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D006487:Hemostasis; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D010973:Platelet Adhesiveness; D010976:Platelet Count; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D013927:Thrombosis / Q000503:physiopathology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D001779:Blood Coagulation Factors; D005338:Fibrin Fibrinogen Degradation Products; D008070:Lipopolysaccharides; D010980:Platelet Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate